Ras-dependent signalling mechanisms have been closely linked with cancer initiation, maintenance, and metastasis. For this reason, elucidation of the mechanisms of Ras regulation is important for the development of novel drug therapies for cancer. One regulator of Ras is CalDAG-GEFIII. This guanine exchange factor facilitates the exchange of GDP for GTP on Ras, thereby leading to Ras activation. Additionally, CaIDAG-GEFIII is able to activate Rap by functioning as a guanine exchange factor for this small G-protein. Currently, very little is known about the regulation/activation of CaIDAG-GEFIII. Our preliminary data shown in this application suggest that diacylglycerol kinase, an enzyme that converts diacylglycerol to phosphatidic acid, down-regulates CalDAG-GEFIII. Thus, the first Specific Aim is to identify which diacylglyerol kinase isoforms are able to regulate CalDAG-GEFIII. Specific Aim #2 is designed to determine how diacylglycerol kinases regulate CalDAG-GEFIII and, subsequently, Rap and Ras signaling pathways. In Specific Aim #3 the physiological role of diacylglycerol kinase zeta on Rap and Ras-dependent signaling pathways will be identified. In this Aim, a knockout mouse will be produced and studied. Together, these approaches will enable the identification of a new mechanism of Ras and Rap-dependent signalling pathway regulation and may pave the way for the development of novel cancer therapies.